Hsp90 is an essential eukaryotic chaperone that helps to produce and maintain the active state of a select set of substrates or clients that are disproportionately linked to signaling processes including many that promote cancer and the emergence of drug resistance in fungi. For these reasons, inhibitor strategies to manipulate the Hsp90 chaperone system promise many potential therapeutic benefits. Inhibitors targeting the ATPase site of Hsp90 effectively limit the emergence of drug resistance in fungi and show promise as anti-cancer agents. However, ATPase inhibitors block all known functions of Hsp90, leading to undesirable side effects. In this proposal, we will determine how thousands of specific perturbations to the Hsp90 chaperone system impact the emergence of drug resistance in fungi and the maturation of three specific clients in order to systematically identify regions of Hsp90 o co-chaperones that could be targeted for the development of client selective inhibitors. To accomplish these goals, we will use the EMPIRIC approach that we developed to determine the effects of all possible point mutations in Hsp90 on yeast growth in the presence and absence of an anti-fungal drug and in combination with knockouts and/or hypomorphic alleles of co-chaperones. We will augment these fitness studies that integrate over the effects of Hsp90 mutants on the function of many clients with analyses of specific clients using yeast reporter strategies. These analyses of environmental and genetic epistasis will provide important mechanistic insights into the network properties of the Hsp90 chaperone system that underlies signaling processes in eukaryotes. In addition, our studies will delineate client and co-chaperone binding sites as well as allosteric sites on Hsp90 that are critical for the maturation o specific clients and for the emergence of drug resistance in fungi.